One objective in the preparation of pharmaceutical solutions, buffer solutions, life support solutions, saline solutions and other such solutions which are to be administered to animals and humans is that they be as free as possible from substances which might cause an adverse reaction in the host. While a goal of zero contamination by substances such as DNA, viruses and endotoxins is always sought, in actual practice very minute amounts of such substances are sometimes present. The Food and Drug Administration (FDA) has set standards for such substances which cannot be exceeded. Manufacturers, ever mindful that a batch of medicant may be rejected if the level of such substances is too high, continually seek new methods to ensure that their products do not exceed FDA standards. Consequently, in all phases of the manufacturing process, manufacturers seek to ensure the purity of the reagents used in the manufacture as well as the final product. Many of the medicants and other products mentioned above are either sold as aqueous solutions or are manufactured in aqueous medium. Consequently, the manufacturers seek to ensure that the water they use is free of DNA, viruses and endotoxins.
One technology that such manufacturers often use is ultrafiltration. U.S. Pat. Nos. 4,431,545 to Pall et al, 4,816,162 to Rosskopf et al, and 4,420,398 to Castino, describe dual-module filtration to remove pathological and/or toxic substances from various fluids including water, blood and plasma. U.S. Pat. No. 4,431,545 utilizes dual filters, one of which has a negative zeta potential and one of which has a positive zeta potential, to filter out positively and negatively charged particles. Neutral particles are removed in accordance with the pore size ratings of the filters which are 0.01 microns or larger as disclosed. U.S. Pat. No. 4,816,162 describes an apparatus that removes immunoglobulins, aIbumin and lipoproteins from blood, blood plasma or serum, but does not describe the removal of DNA or viruses. The filter in this patent is designed for use in circulating and purifying blood during surgery. U.S. Pat. No. 4,420,398 describes a filtration method for separating cell produced antiviral substances, including monoclonal antibodies, from the reaction "broth" in which they are produced. This patent does not indicate whether the resulting species are free of viruses, endotoxins and DNA which may cause a reaction within a patient.
It is known in the prior art that multiple filtration with a 0.04 micron absolute pore size filter will remove viruses of 0.075 micron size, but not smaller viruses. For example, filtration of calf serum containing MS 2 phage (0.024 micron) through 0.04 micron will not remove the virus. In those circumstances where virus can be removed, removal rate is typically 99.9 to 99.99% per filter pass. For example, using a 0.04 micron filter, applicants removed all detectable Reovirus (0.075 micron) from a sample containing 10.sup.8 virus particles per milliliter sample. An article published in the April, 1990 issue of Genetic Engineering News (page 6) commented on the Food and Drug Administration's (FDA) increasing emphasis on viral removal protocols with regard to the preparation of biological pharmaceuticals and the efforts being made by filter manufacturers to achieve higher degrees of virus removal.
Another contaminant which can be present in biological pharmaceuticals such as monoclonal antibodies is DNA. It is generally felt in the industry that the FDA seeks to achieve a DNA level in monoclonal antibody preparations of less than 10 picograms of DNA per dose of monoclonal antibody.
Manufacturers of biological pharmaceutical such as monoclonal antibodies are required to establish Quality Assurance (QA) procedures to which verify that their products meet standards. In the procedures used to show compliance with the standards, it is necessary that the DNA in a sample be concentrated or solid phased (collected in solid form) from a solution of the biological pharmaceutical. It is known that DNA can be concentrated, solid phased or removed from solution by the use of diethylaminoethyl cellulose (DEAE) filter membranes. A manufacturer's literature (Schleicher & Schuell) indicates that DEAE filters will solid phase more than 90% of E. coli DNA from a solution containing 0.2 .mu.g DNA/ml. In a more dilute solution containing 0.001 .mu.g DNA (1 nanogram) more than 80% will be solid phased. The DEAE filters work by binding a protein such as DNA to the filter. However, a major limitation arises in the use of DEAE filters with some monoclonal antibody solutions. For example, it has been found that DNA measurements of monoclonal antibody containing buffer solution having components such as maltose can result in cause false high or low DNA values. In order to assure that the DNA assay values are accurate, these false readings must be eliminated.
Lastly, in addition to viruses and DNA, endotoxins are important contaminating substances in biological pharmaceuticals. While some manufacturers offer column packing materials which are useful in removing endotoxins from protein solutions such as solutions of monoclonal antibodies, such packing materials often result in low product yields after passage of the protein solution through the column. The DEAE filter membranes described above have also been reported to remove endotoxins. However, we have not found the membranes to be effective in removing endotoxins from all sources. In some instances removal is high, whereas in others it is low. This variation is believed to be due to structural variation of the endotoxins themselves in the various samples. The variations in the endotoxins are, in turn, believed dependent on the source of the endotoxin itself and on the chemical treatment it has been subjected to. Having done a careful study of the extant art, we have developed a single filtration device capable of removing virus, DNA and at least some endotoxins to lower levels than previously achieved.